Alternans is a disturbance in normal cardiac rhythm that serves as a clinically important predictor of sudden cardiac death. Although it has been mechanistically linked to the initiation of potentially fatal arrhythmias, the underlying cellular mechanism of alternans itself remains unclear. Action potential (AP) clamping studies have shown that instabilities in either membrane voltage or calcium cycling dynamics can lead to the onset of altemans, but because of the complex interplay between these two systems their individual dynamics are difficult to experimentally assess. Instead, most research focuses on either membrane voltage or (especially) calcium cycling as "the" cause of alternans, negating the probable contribution of both to real myocyte dynamics. Because these two systems represent distinct molecular targets, clinically effective therapy will require an understanding of the role each plays in alternans arrhythmogenesis. The experiments outlined are designed to overcome the inherent difficulties in extrapolating AP clamp study results to normal, undamped cells, and thereby address the relative contributions of voltage and calcium dynamics to alternans in vitro for the first time. To accomplish this, a hybrid computational- experimental approach is proposed, to address the following Specific Aims: 1. Quantification of the sensitivity of cellular alternans to action potential morphology 2. In vitro determination of the contribution of membrane voltage and calcium cycling to cellular alternans. Preliminary studies have confirmed the feasibility of the proposed experiments and show an unequal contribution of both membrane voltage and calcium cycling dynamics to alternans. By determining the true cellular mechanism of cardiac alternans, these aims are directly relevant to the goal of the NHLBI's strategic plan to "delineate mechanisms that relate molecular events to health and disease." Lay summary: Sudden cardiac death (SCD) occurs when the normal rhythm of the heart becomes disordered and is responsible for more than 300,000 deaths each year, in the U.S. alone. Research into the causes of SCD has identified a disorder called "alternans" as a possible precursor to SCD but the cause of alternans itself remains unclear. The experiments proposed will use real heart cells to identify the mechanism(s) of alternans so that we may better try and prevent it and/or its progression to SCD.